Gut-derived endotoxin (LPS), a ligand of the pathogen recognition receptor, Toll-like receptor 4 (TLR4), contributes to alcoholic liver disease (ALD). Ligand engagement of TLR4 activates two signaling pathways: the MyD88-dependent pathway that induces NF- kB and pro-inflammatory gene activation and the MyD88-independent pathway that utilizes the TRIF adaptor, activates IRF3 as a transcription factor to induce Type I interferon production and induces NF-kB activation. Our results suggest that the absence of TLR4 but not of the MyD88 adapter protects mice from ALD. We hypothesize that TLR4-mediated but MyD88-independent signaling pathways which are linked to activation of Type I IFNs and NF-kB-induced inflammatory cytokine genes drive pathology in ALD. We propose that TLR4-induced, TRIF and/or IRF3-mediated signaling is critical in ALD. Reactive oxygen radicals from activation of the NADPH complex contribute to ALD. We hypothesize that TLR4 signaling results in NADPH oxidase complex activation either directly or via MyD88-independent mechanisms. The aims of this proposal are: Aim#1:To assess the role of MyD88-independent TLR4 signaling in ALD by testing liver histology, steatosis and inflammatory markers after chronic alcohol feeding in mice deficient in the TLR4 adapter, TRIF, or IRF3 to evaluate whether the alcohol-induced MyD88- independent pathway activation occurs downstream of IRF3. IFNA 1/2 receptor deficient mice will be tested to assess the involvement of IRF3-induced Type I interferons in alcoholic liver injury. Aim #2: To evaluate signaling mechanisms involved in amplification of MyD88-independent TLR4- signaling elements in alcoholic liver disease by: a) exploring interactions between TLR4 signaling and NADPH oxidase components in the liver and in isolated Kupffer cells; b) investigating the interaction between NF-kB and IRF signaling pathways using an inhibitor of IKK? (NEMO adapter) in vivo; c) evaluating the role of TNF? in maintaining an IFN?-mediated autocrine loop in the sustained increase in inflammatory gene expression in the liver and Kupffer cells. Aim #3:To determine the cell type specificity of TLR4-mediated signals between hepatocytes and bone marrow-derived inflammatory cells in alcoholic liver injury in bone marrow chimera mice including wild-type mice with TLR4-,TRIF- or IRF3-deficient bone marrow and in TLR4-,TRIF- or IRF3- deficient mice with wild-type bone marrow. These experiments will provide novel insights into the TLR4-mediated signaling in alcoholic liver injury. Identification of the specific elements and cell-specificity of the TLR4 signaling pathways in meditation of alcoholic liver disease will provide novel therapeutic targets for future drug development. PUBLIC HEALTH RELEVANCE: Gut-derived endotoxin (LPS), a ligand of the pathogen recognition receptor, Toll-like receptor 4 (TLR4), contributes to alcoholic liver disease. Ligand engagement of TLR4 activates two signaling pathways: the MyD88-dependent pathway that induces NF-kB and pro-inflammatory gene activation and the MyD88-independent pathway that utilizes the TRIF adaptor, to induce Type I interferon production and induces NF-kB activation. Our results suggest that the absence of TLR4 but not of the MyD88 adaptor protects mice from alcoholic liver disease.